Leishmania is a parasite that causes severe skin ulcers or visceral disease in people living in the developing world. Every year, it infects millions of people, and kills over 50,000. Drugs for treating infection by Leishmania are very toxic, and the parasite is developing resistance to them. For these reasons, new drugs are needed to treat infections by this parasite. Leishmania must live inside macrophages to survive in and cause disease within a host. To enter macrophages, Leishmania binds to integrin receptors on the cell surface. The parasite then triggers its own uptake through the process of phagocytosis. The mechanisms that permit cell entry by Leishmania are not well understood. This project aims to characterize the signaling events that translate the binding of cell surface receptors to the reorganization of the cytoskeleton, which is required for internalization of Leishmania. Understanding the mechanisms of cell entry by Leishmania will help explain how this parasite causes disease. Furthermore, if one could disrupt the pathways that lead to invasion of macrophages by Leishmania, the parasite should not be able to survive within a host to cause disease. Therefore, these studies may also suggest new drugs to treat this parasitic infection. Abl family kinases are proteins that transfer signals from extracellular integrin receptors to the cytoskeleton in order to orchestrate cell movement. These kinases are known to activate cortactin, which triggers actin-based cell edge protrusions. HS1 (hematopoietic lineage cell-specific protein 1) is a protein related to cortactin that is found in macrophages. Preliminary work in the laboratory demonstrates that signals from cell surface receptors activate Abl family kinases and stimulate phagocytosis. However, signaling pathways requiring Abl family kinases or HS1 have not been linked to uptake of Leishmania by macrophages. The central hypothesis in this proposal is that Leishmania uses a signaling pathway that requires integrin receptors, Abl family kinases, and HS1 to enter cells and cause disease. Specific Aim 1 addresses whether Abl family kinases mediate cell entry and infection by Leishmania, and if 22 integrins directly bind and activate Abl family kinases. Specific Aim 2 addresses whether Leishmania requires HS1 signaling for cell entry and infection;it also tests whether Abl family kinases activate HS1. In both specific aims, macrophages will be infected with Leishmania to understand the requirement for each of these signaling components for cell entry, and mice will be infected with Leishmania to determine the significance of these proteins in disease. Biochemical assays with purified proteins and molecular biology techniques to disrupt interactions between proteins will also be employed to elucidate the interfaces between key components in this signaling pathway. By identifying whether signaling though integrins, Abl family kinases, and HS1 allows Leishmania to enter cells and cause disease in mice, our understanding of the process of Leishmania infection will improve. PUBLIC HEALTH RELEVANCE: This project seeks to explain how a human parasite called Leishmania gains entry into cells in order to survive. Leishmania infects millions of people every year worldwide, and kills more than 50,000 of them. We hope that if we understand how Leishmania enters cells, new drugs can be developed that block cell entry, and therefore, disease caused by this parasite.